Title

Author

Date of Graduation

5-2009

Document Type

Thesis

Degree Name

Bachelor of Science in Biological Engineering

Degree Level

Undergraduate

Department

Biological and Agricultural Engineering

Advisor

Ye, Kaiming

Abstract

The detection of viable Escherichia coli (E. coli) by the bacteriophage-based fluorescent technique using the enhanced green fluorescent protein (EGFP) was tested in this project. EGFP, a mutant of green fluorescent protein (GFP), was used due to its high fluorescent efficiency compared to wild-type GFP. First, E. coli bacteria were cultured to the mid-exponential phase in the 2Ã— YT medium and then they were infected with the M13 bacteriophages containing the EGFP gene. Infected bacteria were incubated for different time intervals, and the expression of EGFP on the E. coli membrane was tested by the PerkinElmer LS 55 Luminescence Spectrometer, which measured the relative fluorescence intensity using the emission spectrum technique. The expected emission maximum wavelength for pure EGFP was around 510nm; however, the detected emission maximum wavelength in this project was around 525nm. The emission maximum wavelength of the crude sample in this project was red-shifted about 15nm compared to that of the standard pure EGFP sample. It could be concluded that further mutation of the bacteriophages produced the protein with the longest emission maximum wavelength or yellow fluorescent protein (YFP). Optimization in each intermediate step might help reduce errors or help prevent unwanted mutation to ultimately reduce the red-shift, and hence bring the emission maximum wavelength back to the desired one. It could also be concluded that the cellular autofluorescence from endogenous flavin such as FAD was predominant in the E. coli cells. However, cellular autofluorescence problem must be handled, for example, by purifying the sample, using the bandpass fluorescence filter, or by combining with immunofluorescence technique. Key words: E. coli TG1, bacteriophage, fluorescent technique, GFP, EGFP, red-shift, and cellular autofluorescence.